【作者】 段炼；

【导师】 杨斌盛；

【作者基本信息】 山西大学 ， 无机化学， 2010， 博士

【摘要】 中心蛋白(Centrin)是属于EF-hand钙结合蛋白超家族的分子量较小(约20kD)的酸性蛋白。最初中心蛋白是作为鞭毛单细胞生物中连接核和鞭毛器的纤维的主要成分被鉴定得到的,随后发现中心蛋白是中心粒,中心体和有丝分裂纺锤体极体中一种广泛存在的成分,与细胞的分裂,细胞中中心体的定位、定向,纺锤体极体分离,微管切除,纤维系统的收缩等有关。其中基于中心蛋白的纤维系统的收缩现象,与在Ca2+存在下,不论是酵母,藻类或是人类的中心蛋白都会形成多聚体密切相关。八肋游仆虫中心蛋白(EoCen)是从一种在进化上处于特殊地位的单细胞真核生物——八肋游仆虫中克隆得到的,将其作为研究金属离子(Ca2+、Ln3+)进入体内的靶蛋白模型,对于阐明稀土生物效应的分子基础具有重要的意义。EoCen共含有168个氨基酸残基,与人中心蛋白1(HsCen1),人中心蛋白2(HsCen2)和人中心蛋白3(HsCen3)的序列同源性分别为60%,62%和66%,与研究最为广泛的EF-hand结构蛋白钙调蛋白(CaM)的同源性为50%。最近,八肋游仆虫中心蛋白N端结构域的NMR结构刚刚被报道(2joj. pdb)。与CaM一样,EoCen由两个相互独立的结构域组成,中间由一段弹性螺旋相连。每个结构域都由一对螺旋—环—螺旋结构的EF-hand结构基元组成,每个结构基元能够结合一个Ca2+。与其他物种的中心蛋白一样,在Ca2+存在下,EoCen也会形成多聚体,这一点与CaM明显不同。本文将着重研究金属离子诱导下八肋游仆虫中心蛋白在溶液中的聚集性质。本文用定点突变的方法构建得到了九个酪氨酸突变体(Y46F, Y72F, Y79F, N-Y46F, N-Y72F, N-Y79F, N-Tyr46, N-Tyr72, N-Tyr79)重组质粒。通过DNA序列分析确定所得到的突变体只含有目的位点的突变基因,而不包含其它任何位点的随机突变。将重组质粒转入大肠杆菌BL21(DE3)细胞中,在诱导剂IPTG诱导下实现蛋白可溶性表达。融合蛋白经PPase酶切和GST亲和层析之后得到目的蛋白。另外,将野生型蛋白(EoCen)和本课题组之前构建完成的蛋白片段分子(N-EoCen, C-EoCen,Δ23EoCen,P23,P123)诱导表达纯化得到目的蛋白。经SDS-PAGE分析显示所有蛋白均不含杂蛋白条带,达到实验所需纯度。首先对金属离子诱导下的EoCen的聚集性质进行了系统的分析表征。含有不同结构域的蛋白片段分子(EoCen, N-EoCen, C-EoCen,Δ23EoCen,P23,P123)的化学交联实验表明蛋白的N末端是聚集发生的主要部位。天然胶电泳显示在Lu3+存在下,只有完整的野生型蛋白分子能够显示出多聚体条带。用共振光散射(RLS)的方法分析了多种理化因素,包括温度,蛋白浓度,离子强度,酸度对Lu3+诱导的EoCen聚集性质的影响。不同金属离子与蛋白结合的对照实验表明,Lu3+或Ca2+在EoCen N端的结合有利于蛋白的聚集,且Lu3+表现出更强的促进效应,而Mg2+的结合不能起到相同或相似的作用。2-ptoluidinylnaphthalene-6-sulfonate (TNS)结合实验以及离子强度实验证明Lu3+结合诱导的EoCen的聚集与蛋白疏水区的暴露密切相关。酸度效应实验中,不同片段分子蛋白的对照研究表明静电作用在EoCen的聚集过程中影响较小。酪氨酸残基是组成蛋白——蛋白相互作用的“热点”区域的主要氨基酸残基之一,且其具备发光性质。本课题组之前的工作以Tb3+作为荧光探针证明EoCen存在4个Ca2+结合位点,且Tb3+敏化荧光的增强主要来源于EoCen的N末端所含的三个酪氨酸残基(Tyr-46,Tyr-72,Tyr-79)与结合的Tb3+之间的能量转移。为了找到EoCen聚集时的蛋白——蛋白相互作用过程中,以及EoCen与Tb3+结合时的能量转移过程中起重要作用的酪氨酸残基,本文用定点突变的方法得到了九个分别含有一个或两个酪氨酸残基的EoCen突变体。实验结果显示79位酪氨酸的突变对荧光敏化的影响最为明显,使得Tb3+的荧光敏化强度降低达到50%之多。Forster能量转移机理得出79位酪氨酸与蛋白N端结合的两个Tb3+之间的距离最近(8.8±0.2A)。稳态荧光及时间分辨荧光参数的测定表明处于疏水环境中的Tyr-79量子产率最高(3.27×10-2),荧光寿命相对较长(2.20 ns),且平均寿命数据的标准偏差(σ=0.271 ns)最小。另外,分子模拟显示Tyr-79的酚羟基与Asn-39的侧链上的氧原子之间形成了重要的氢键。酪氨酸与Tb3+的荧光动力学实验证明酪氨酸荧光的猝灭绝大部分来源于EoCen在金属离子结合诱导下产生的聚集,研究发现不同酪氨酸突变体所表现出的荧光猝灭程度有所不同。以全分子单酪氨酸突变体(Y46F, Y72F, Y79F)为研究对象,经RLS和化学交联分析显示,Tyr-79是蛋白聚集过程中贡献最大的酪氨酸残基。细胞渗透型小分子抑制剂Monastrol通常被认为能够特异性地抑制驱动蛋白Eg5的活性,导致细胞有丝分裂停滞和单极纺锤体的形成,从而表现出明显的抗肿瘤活性。中心蛋白作为一种附着于中心体上的广泛存在的蛋白,在中心体复制过程中发挥着重要的作用。已有报道称中心体的大量增殖很可能与癌症有关。本文研究发现Monastrol在体内能够有效抑制转化有pGEX-6p-1-EoCen重组质粒的大肠杆菌细胞的生长,以及八肋游仆虫的增殖分裂；在体外能够抑制Lu3+诱导的EoCen的聚集。合成得到的两个Monastrol类似物(Compound 1和Compound 2)不能达到相同的抑制效果。荧光滴定实验表明1个EoCen蛋白分子能够结合4个Monastrol分子,并且得到不同温度下EoCen与Monastrol相互作用的结合常数。热力学数据分析显示EoCen与Monastrol结合的主导作用力为疏水作用力。Monastrol对蛋白聚集抑制的程度与金属离子结合所诱导的蛋白疏水区的暴露密切相关。蜂毒素(Melittin)是一种从蜂毒中提取出的由26个氨基酸残基构成的两亲性小分子肽,被广泛用作研究蛋白——蛋白相互作用或者蛋白——脂类相互作用的工具。本文研究表明EoCen与Melittin主要以静电力方式相互作用。初步证明Melittin在EoCen上的结合对Lu3+诱导的EoCen的聚集有抑制作用。中心蛋白的两大作用机制——纤维收缩和信号转导可能存在着共同的分子基础。更多还原

【Abstract】 Centrin is an acidic, low molecular weight (Mr～20000) protein that belongs to the EF-hand superfamily of calcium binding proteins. This calcium-binding protein (centrin) was first identified as a major component of the fibers that link the nucleus to the flagellar apparatus in flagellated unicells, and later, it was shown to be a ubiquitous component of centrioles, centrosomes, and mitotic spindle poles. Cenrin is required for the cell division, centrosome positioning and orientation, mitotic spindle segregation, as well as microtubule severing, and plays an essential role in contraction of centrin-based fiber systems in eukaryotic cells. A distinguishing feature of centrin-based fiber systems is that they all show calcium-induced contractile behavior, which is closely related to the fact that centrins from yeast (Cdc31p), algae (Scherffelia dubia centrin (SdCen)), or humans (HsCen1, HsCen2) may form multimers in the presence of Ca2+.Ciliate Euplotes octocarinatus centrin (EoCen) is first reported by our laboratory, which is cloned from Euplotes octocarinatus, a unicellular eukaryotic protozoon located in a special phylogenic degree. Using EoCen as a model of metal ion-binding target is significant for elucidating the molecular basis of biological effects of rare earth elements. EoCen is a protein of 168 residues, which shares about 60,62 and 66% sequence identity with human centrin 1, human centrin 2 and human centrin 3, respectively, and shares approximately 50% sequence identity with the well studied EF-hand protein calmodulin (CaM). Recently, the structure of the N-terminal domain of EoCen was obtained by NMR (2joj. pdb). Like CaM, centrin consists of two independent domains tethered by a flexible linker, each domain comprising a pair of EF-hand motifs of helix-loop-helix that can potentially bind two calcium ions. EoCen may form multimers in the presence of Ca2+ as same as centrins from other species, in clear contrast with CaM. This dissertation focuses on the metal ion-dependent self-assembly of EoCen. In the present study, nine mutants (Y46F, Y72F, Y79F, N-Y46F, N-Y72F, N-Y79F, N-Tyr46, N-Tyr72 and N-Tyr79) were first obtained by site-directed mutangenesis. In all cases, the presence of the specific mutation and the lack of random mutations were verified by DNA sequence analysis, which was conducted by a commercial company. Recombinant plasmids were transformed in E.coli BL21 (DE3), then the fusion expression of the mutants were performed by the induction of IPTG Fusion protein was cut by PPase and was purified by GST affinity chromatography. At the same time, the wild-type EoCen and the truncated form of it (EoCen, N-EoCen, C-EoCen,Δ23EoCen, P23, and P123), which are previously constructed by our group, were expressed and purified. The final product is examined by SDS-PAGE, and all of the protein products were of high purity.In order to systematically characterize the metal-ion dependent self-assembly of EoCen, we initiated a physicochemical study of the self-assembly properties of the purified protein (EoCen, N-EoCen, C-EoCen,Δ23EoCen, P23, and P123) in vitro. The critical role of the N-terminal domain of EoCen played in the self-assembly was exhibited by chemical cross-linking experiment. The native PAGE results indicate that only the integral protein shows multimers in the presence of Lu3+. The dependence of Lu3+-induced self-assembly of EoCen on various chemical and physical factors, including temperature, protein concentration, ionic strength and pH, was characterized using resonance light scattering (RLS). Control experiments with different metal ions suggest that Ca2+and Lu3+binding to the N-terminal domain of EoCen are all positive to the self-assembly of the protein, and Lu3+ exhibit the stronger effect, however, Mg2+ alone cannot take the same effect. The experiments of 2-ptoluidinylnaphthalene-6-sulfonate (TNS) binding and ionic strength effect demonstrate that the lutetium(Ⅲ)-dependent self-assembly is closely related to the exposure of hydrophobic cavity. Control experiment on pH value with EoCen and the fragments of it, N-terminal domain of EoCen (N-EoCen), indicates that the electrostatic effect is of small tendency to be served as the main driving force in the self-assembly of EoCen.Tyrosine is one of the most preferred residues in "hot spots" of protein-protein interactions. In addition, tyrosine is of fluorescence properties. It has been proven that EoCen has four calcium-binding sites by using Tb3+ as fluorescence probe. The sensitized emission arises from a nonradiative energy transfer between the three tyrosine residues (Tyr-46, Tyr-72, and Tyr-79) of N-terminal half and the bound Tb3+ions. In addition, To find the most critical one of the three tyrosine residues in the process of fluorescence resonance energy transfer (FRET) and in the protein self-assembly, nine mutants (Y46F, Y72F, Y79F, N-Y46F, N-Y72F, N-Y79F, N-Tyr46, N-Tyr72 and N-Tyr79) of EoCen, which contain one or two tyrosine residues were obtained by site-directed mutagenesis. The aromatic residue-sensitized Tb3+ fluorescence of N-Y79F was mostly affected, which displayed a 50% reduction compared with wild-type N-EoCen. Tyr-79 is of the nearest mean distance (8.8±0.2A) of protein bound Tb3+(at sitesⅠ/Ⅱ), which is calculated via the Forster mechanism. The steady-state and time-resolved fluorescence parameters of the wild-type N-EoCen and the three double mutants suggest that Tyr-79, which exists in a hydrophobic environment, has the highest quantum yield (3.27×10-2) and a relatively long average lifetime (2.20 ns). The standard deviation of the average lifetime of Tyr-79 (σ=0.271 ns) is the least among the three tyrosine residues. In addition, molecular modeling shows that a critical hydrogen bond is formed between the 4-hydroxyl group of Tyr-79 and the oxygen from the side chains of residue Asn-39. Kinetic experiments of tyrosine and Tb3+fluorescence demonstrate that the tyrosine fluorescence quenching is largely originated from the self-assembly of EoCen, and that the quenching degrees of the mutants differ from one another. Resonance light scattering (RLS) and cross-linking analysis carried out on the full-length single mutants (Y46F, Y72F, and Y79F) showed that Tyr-79 also plays the most important role in the Tb3+-dependent self-assembly of EoCen among the three tyrosines.Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibit antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells (pGEX-6p-1-EoCen) and the division of the Euplotes octocarinatus cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compound 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.Melittin, a basic 26-amino acid residue amphiphilic peptide from bee venom, is widely used as a tool for the study of protein-lipid and protein-protein interactions in artificial and biological membranes. In the present study, we proved that the main acting force in the binding of melittin to EoCen is the electrostastic force. Evidence that melittin binding of EoCen have inhibited the Lu3+-induced self-assembly of EoCen is preliminary. The two action mode of centrin may underlie the same molecular mechanism.更多还原